The anti‐parasitic drug miltefosine suppresses activation of human eosinophils and ameliorates allergic inflammation in mice

Background and Purpose Miltefosine is an alkylphosphocholine drug with proven effectiveness against various types of parasites and cancer cells. Miltefosine is not only able to induce direct parasite killing but also modulates host immunity, for example by reducing the severity of allergies in patients. To date, there are no reports on the effect of miltefosine on eosinophils, central effector cells involved in allergic inflammation. Experimental Approach We tested the effect of miltefosine on the activation of human eosinophils and their effector responses in vitro and in mouse models of eosinophilic migration and ovalbumin‐induced allergic lung inflammation. Key Results The addition of miltefosine suppressed several eosinophilic effector reactions such as CD11b up‐regulation, degranulation, chemotaxis and downstream signalling. Miltefosine significantly reduced the infiltration of immune cells into the respiratory tract of mice in an allergic cell recruitment model. Finally, in a model of allergic inflammation, treatment with miltefosine resulted in an improvement of lung function parameters. Conclusion and Implications Our observations suggest a strong modulatory activity of miltefosine in the regulation of eosinophilic inflammation in vitro and in vivo. Our data underline the potential efficacy of miltefosine in the treatment of allergic diseases and other eosinophil‐associated disorders and may raise important questions regarding the immunomodulatory effect of miltefosine in patients treated for leishmania infections.


| INTRODUCTION
To date, miltefosine (Impavido®) is the only oral drug approved for the treatment of leishmaniasis with limited mild or moderate side effects (Pijpers et al., 2019). The development of miltefosine is a success story of public-private partnership, a breakthrough in medicine affordability and patient drug adherence, landing it on the World Health Organization (WHO)'s List of Essential Medicines (Berger et al., 2017;Sunyoto et al., 2018). Miltefosine disrupts membrane structures and affects phosphatidylcholine synthesis in susceptible promastigote cells (Pinto-Martinez et al., 2018;Rakotomanga et al., 2007). Due to its detergentlike properties, miltefosine is thought to interact with the mucosa of the gastrointestinal tract during oral use and cause its most commonly listed side effects-nausea, vomiting and diarrhoea (Bhattacharya et al., 2007). During prolonged treatment, the severity of the side effects was reported to decrease over time (8.2% during Week 1 to 3.2% during Week 4) (Bhattacharya et al., 2007).
Miltefosine exerts immunomodulatory effects on human cancer cells by inhibiting the PI3K/Akt signalling pathway (Ruiter et al., 2003), induces IL-12-dependent Th1 responses (Wadhone et al., 2009) and shows anti-inflammatory effects in endothelial cells, suppressing vascular inflammation (Fang et al., 2019). However, the immunomodulatory effects of miltefosine on primary human cells have so far only been described for T cells (Bäumer et al., 2010) and mast cells (Weller et al., 2009).
Miltefosine increases membrane fluidity (Moreira et al., 2014), modulates lipid raft-dependent signalling (Weller et al., 2009) and could therefore be an attractive drug candidate for the treatment of diseases characterized by abundant lipid raft activation, such as allergic diseases (Dölle et al., 2010). Miltefosine attenuates allergic inflammation in T cell-dependent mouse models of dermal inflammation (Bäumer et al., 2010), improves local dermatitis in patients with atopic dermatitis (Dölle et al., 2010), inhibits activation and degranulation of mast cells, and significantly reduces allergic disease manifestation in patients Maurer et al., 2013;Rubíková et al., 2018).
Surprisingly, there are no reports on the effects of miltefosine on eosinophils, a key cell type involved in the initiation and propagation of immune responses in allergic diseases (Stone et al., 2010). Here, we studied in detail whether miltefosine exerts immunomodulatory effects on eosinophils in vitro and in mouse models of allergic lung inflammation. Fixative solution was prepared by adding 9 ml of distilled water and 30 ml of FACS sheath fluid (BD Biosciences) to 1 ml of CellFix (BD Biosciences, Vienna, Austria) as described previously (Knuplez, Curcic, et al., 2020).

What is already known
• Miltefosine is an orphan drug marketed for the treatment of leishmaniasis.
• Miltefosine reduces the severity of allergies in patients.

What this study adds
• Miltefosine inhibits activation of human eosinophils and suppresses human eosinophil effector responses.
• Miltefosine inhibits the infiltration of immune cells in the airways and improves animal lung function.
What is the clinical significance • Miltefosine may serve as a potential candidate for the treatment of eosinophil-related diseases.
• Miltefosine treatment may influence eosinophil host responses in leishmania-infected patients.

| Mice
Animal studies are reported in compliance with the ARRIVE guidelines (Percie du Sert et al., 2020) and with the recommendations made by the British Journal of Pharmacology (Lilley et al., 2020) where there were randomly divided in three groups (negative control-vehicle; positive control-ovalbumin or eotaxin stimulated and miltefosine pretreated and ovalbumin or eotaxin stimulated group). Experiments, where bronchoalveolar lavage fluid was collected, could not be performed blinded, due to investigator treating the mice prior to fluid collection. Lung function testing was performed blinded, since Investigator 1 treated the mice and Investigator 2 independently performed lung function testing on mice in a random order.
For all animal experiments, at least five mice were included in each group and at least two repeat experiments were carried out.
Experiments were designed to make sample sizes relatively equal and randomized among comparison groups. Sample sizes were determined according to previous studies with similar analyses (Knuplez, Curcic, et al., 2020;Theiler et al., 2019).

| Blood sampling and eosinophil isolation
Blood sampling from healthy volunteers was approved by the Institutional Review Board of the Medical University of Graz (17-291 ex 05/06). All participants signed a written informed consent.
Firstly, platelet-rich plasma was removed by centrifugation. Next, red blood cells and platelets were removed by dextran sedimentation and polymorphonuclear leukocytes preparations were obtained by density gradient separation. Eosinophils were isolated from polymorphonuclear leukocytes by negative magnetic selection using a cocktail of biotin-conjugated antibodies against CD2, CD14, CD16, CD19, CD56 (neural cell adhesion molecule 1), CD123 (interleukin 3 receptor, α subunit) and CD235a (glycophorin A) as well as Anti-Biotin Micro-Beads from Miltenyi Biotec (Bergisch Gladbach, Germany). Eosinophil purity was determined by morphological analysis of Kimura-stained cells and was typically greater than 97%.

| Flow cytometric analysis of intracellular kinase phosphorylation
Isolated eosinophils were pretreated with either vehicle or miltefosine 20 (μM) (15 min, RT). Following the pretreatment, cells were incubated with 10-nM eotaxin-1 (CCL11) (3 min, 37 C). Subsequently, cells were fixed, permeabilized and stained as described previously (Knuplez, Curcic, et al., 2020). Phosphorylation of Akt residues in fixed eosinophils was quantified as the increase of fluorescence in the FITC fluorescence channel from unstimulated control.

| In vivo chemotaxis
In vivo eosinophil migration was induced by intranasal application of 4-μg eotaxin-2 CCL24 in 8-week-old male and female heterozygous IL-5 transgenic (IL-5Tg) mice (BALB/c background). The mice and their littermate controls received oral gavages of miltefosine (20 mgÁkg −1 in 0.9% NaCl) or vehicle for three consecutive days before CCL24 application. Bronchoalveolar lavage fluid was collected 4 h after experiment had started. Migration of eosinophils was evaluated by flow cytometric counting of highly granular (high side scatter) CD11c − / Siglec-F + cells, as described previously (Knuplez, Curcic, et al., 2020).

| Corticosterone measurement in plasma
Corticosterone levels were assessed in plasma of BALB/c mice treated with oral gavages of miltefosine (20 mgÁkg −1 ) once daily for 3 days. A blood sample was collected via cheek bleed 5 h after first miltefosine application on Day 1, as well as 4 h after last treatment on Day 3. Corticosterone levels were determined with a specific enzyme immunoassay kit (Assay Designs, Ann Arbor, MI, USA) with a sensitivity of 0.027 ngÁml −1 as previously described (Farzi et al., 2015) and

| Statistical analysis
The data and statistical analysis comply with the recommendations of the British Journal of Pharmacology on experimental design and analysis in pharmacology (Curtis et al., 2018). Statistical analysis was performed using the GraphPad Prism™ 6 software (GraphPad Software, Inc., CA, USA). Data were normalized to baseline (1 or 100%) of the means of negative control in experiments performed with eosinophils isolated from human donors to reduce interindividual source of variation.
Statistical analysis was only performed for groups where n ≥ 5.
Additional preliminary data (n = 3) on p-Akt phosphorylation in eosinophils were included in the manuscript to suggest a mechanism previously shown for other cell types (Chugh et al., 2008;Ruiter et al., 2003). The group size given for each experiment is the number of independent values (individual human eosinophil donors or mice). Statistical analysis was performed using these independent values.
Data were tested for normality using D'Agostino and Pearson omnibus normality test. If normality was assumed, comparisons among multiple groups were performed with one-way ANOVA or two-way ANOVA. For these analyses, post hoc pairwise comparisons were performed using Bonferroni's multiple comparison test (or Dunnett's multiple comparison test, when comparing samples to the control group), only if a main effect for at least one factor or the interaction between two factors showed statistical significance and if there was no significant variance in homogeneity. Cytokine levels were compared using Mann-Whitney U test. Significance level for the analyses was set to α = 0.05 and significant differences are indicated with the corresponding P value, *P ≤ 0.05.

| Miltefosine suppresses eosinophil activation in vitro
First, we tested the viability of eosinophils after pretreatment with different concentrations of miltefosine. Importantly, miltefosine (up to 20 μM, in the presence of 1-mgÁml −1 bovine serum albumin) showed no toxic effects on eosinophils ( Figure S1).
During the state of allergic inflammation, elevated concentrations of cytokines and chemoattractants in the blood activate eosinophils, which leads to a rearrangement of their actin filaments (the so-called "shape change") (Willetts et al., 2014) and results in an up-regulation of the adhesion molecules integrins (e.g., CD11b/CD18 and Mac-1) on the cell surface (Jia et al., 1999). When human eosinophils were pretreated with miltefosine, we could observe a statistically significant inhibition of their shape change (by approx. 50%) induced by CCL11 stimulation (Figure 1a,b) when using the highest concentration of miltefosine (20 μM). Miltefosine addition did not alter eosinophil shape change in the absence of external stimuli ( Figure S2). When isolated eosinophils were pretreated with 20-μM miltefosine, up-regulation of CD11b was reduced by about 50% (Figure 1c,d).
To determine whether miltefosine has an effect on the chemotaxis of human eosinophils, we performed in vitro chemotaxis assays

| Miltefosine ameliorates ovalbumin-induced lung inflammation
Next, we investigated whether the in vitro results obtained with isolated human eosinophils are also relevant in vivo. We first performed Ca 2+ flux assays using mouse bone marrow-derived eosinophils to test whether mouse eosinophils behave similar to human-isolated eosinophils (Figure 4a Next, we performed an in vivo eosinophilic migration test using IL-5Tg mice. This strain of mice is characterized by eosinophilia due to increased production of IL-5. Together, intranasal eotaxin application in IL-5-primed eosinophils results in abundant and eosinophil accumulation in the bronchoalveolar lavage fluid and lungs of animals (Ochkur et al., 2007). We treated IL-5Tg mice for three consecutive days perorally with miltefosine (20 mgÁkg −1 ) (Figure 4c). We used a dosing regimen comparable with other studies in mice testing miltefosine  Figure S5A); however, when BALB/c mice were treated with miltefosine, no increase in neutrophils was observed ( Figure S5B). By testing plasma of BALB/c mice for their corticosterone levels, we observed no significant differences at both of the two tested time points (Figure S6A,B).
We next tested the efficacy of miltefosine in an acute model of allergic lung inflammation. Ovalbumin was used as a model allergen to reproduce key features of clinical asthma, such as airway hyperresponsiveness to methacholine (Kumar et al., 2008). The treatment protocol of the model is shown in Figure 5a. We observed that daily peroral treatment with miltefosine markedly reduced the number of several infiltrating immune cells into airways of ovalbumin-challenged wild-type mice. Flow cytometric analysis of the composition of immune cells showed that the number of eosinophils as well as infiltrating T cells, B cells and dendritic cells was reduced by 50% upon miltefosine treatment (Figure 5b). Of note, mice treated with miltefosine showed significantly improved lung resistance and a trend towards improved lung compliance (Figure 5c). In order to test whether a decrease in eosinophil numbers was responsible for the reduction of other immune cells, eosinophil-deficient (Δdbl GATA-1) mice were exposed to the same ovalbumin-induced allergic model. In

| DISCUSSION
In the present study, we show for the first time that the Food and Drug Administration (FDA)-approved drug miltefosine inhibits the The effects of miltefosine have previously been studied on some other immune cells. Notably, miltefosine was found to inhibit degranulation and antigen-induced chemotaxis of mast cells by modulating lipid rafts and by inhibiting cytosolic PKC (Rubíková et al., 2018). In contrast to our findings with eosinophils, calcium flux in mast cells was apparently not affected by miltefosine pretreatment, indicating cell type-specific differences. However, similar to mast cells, miltefosine led to an inhibition of effector functions and mediator release in eosinophils. In macrophages, miltefosine was found to  (Iacano et al., 2019). Given the fact that TLR-4 stimulation on eosinophils can help polarize macrophages towards pro-or antiinflammatory phenotypes (Yoon et al., 2019), this finding further supports the evidence that miltefosine may influence the interplay and balance between various immune cell types during the state of inflammation.
It is noteworthy that in all our in vitro experiments, non-toxic concentrations of miltefosine were used to distinguish our results from the non-specific cytolytic effects of the drug. In particular, since homeostatic functions such as tissue remodelling and plasma cell survival (Jacobsen et al., 2012) have recently been attributed to eosinophils, we were mainly interested in inhibiting eosinophil overactivation, to prevent their potential tissue-damaging effector functions. For our in vivo experiments, we used a dosage regimen, comparable with other studies in mice testing miltefosine (Bäumer et al., 2010). Δdbl GATA-1 mice. We discovered that the decreased infiltration of most immune cells was at least partially due to the decreased eosinophil numbers. This is not unexpected, since activated eosinophils are known to attract and activate other immune cell types such as neutrophils (Yousefi et al., 1995) or B cells (Chu et al., 2011). Moreover, eosinophil-derived CCL17 and CCL22 have proven to be crucial in attracting effector T cells in localized allergic inflammation (Jacobsen et al., 2008). Interestingly, we observed a decrease in dendritic cell since it was discovered that eosinophil-derived IFN-γ induces airway hyperresponsiveness and lung inflammation even in the absence of lymphocytes (Kanda et al., 2009). Interestingly, IFN-γ was also found to up-regulate several eosinophil effector functions (Ishihara et al., 1997;Takaku et al., 2011) and promote their survival (Fujisawa et al., 1994).
When we examined the composition of immune cells in mouse blood, miltefosine-treated and CCL24-stimulated IL-5Tg animals showed an increased neutrophil count, yet miltefosine-treated BALB/c animals showed no altered neutrophil numbers at baseline. A previous study showed that patients treated with miltefosine exhibited increased levels of the neutrophilic chemokine IL-8 (CXCL8) (Mukhopadhyay et al., 2011. This finding remains to be confirmed in mice. Increased corticosterone levels in mice induced by miltefosine could be another plausible explanation for both increased neutrophil numbers (Liles et al., 1995) and decreased airway inflammation (Suqin et al., 2009). Furthermore, an inverse association between endogenous glucocorticoid and IFN-γ levels was observed in allergic lung inflammation (Suqin et al., 2009). Nonetheless, we observed no significant alterations in corticosterone levels in miltefosinetreated mice. one of the primary cells recruited to the sites of leishmania infection (de Oliveira Cardoso et al., 2010) and have been shown to help control parasite load (Watanabe et al., 2004) in mice, it might be of interest to further investigate this issue in patients treated with miltefosine. In line with the present study, we have previously shown that saturated lysophosphatidylcholines, which are structurally similar to miltefosine, inhibit eosinophil effector responses (Knuplez, Curcic, et al., 2020;Knuplez, Krier-Burris, et al., 2020;Trieb et al., 2019).
A limitation of our work needs to be noted. Ovalbumin was used as a model allergen in our in vivo studies, albeit this model fails to completely reflect the aetiology of human asthma and its multi-step developmental process, including environmental factors associated with the disease. Further experiments with other physiological relevant antigens are needed to validate the relevance of our data in human disease setting.
In summary, we demonstrate the inhibitory effect of the orphan drug miltefosine on human eosinophils and its anti-inflammatory effect in vivo in a model of allergic inflammation. Our data highlight the potential efficacy of miltefosine or related molecules in the treatment of allergic diseases and other eosinophil-associated disorders.

FUNDING INFORMATION
This study was supported by the Austrian Science Fund (FWF Grants

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request. Some data may not be made available because of privacy or ethical restrictions.